CN110144363B

CN110144363B - Insect-resistant herbicide tolerant corn transformation events

Info

Publication number: CN110144363B
Application number: CN201810140851.4A
Authority: CN
Inventors: 刘博林; 佘秋明; 谭超; 许洁婷; 王绪霞; 田裴秀子; 聂东明; 韩宇; 马崇烈; 章旺根
Original assignee: China National Seed Group Co Ltd
Current assignee: China National Seed Group Co Ltd
Priority date: 2018-02-11
Filing date: 2018-02-11
Publication date: 2022-11-18
Anticipated expiration: 2038-02-11
Also published as: US11479790B2; CN110144363A; US20210054402A1; WO2019154373A1; BR112020014816A2

Abstract

The present application provides insect-resistant herbicide tolerant corn transformation events, and provides related development methods, detection methods, and applications. The method comprises the following steps of taking a maize inbred line 249 as a receptor, and carrying out agrobacterium-mediated genetic transformation to obtain a maize plant in which an exogenous gene insert is inserted into a specific genome site, wherein the exogenous gene insert comprises the following three genes: insect-resistant gene, glufosinate-resistant gene and glyphosate-resistant gene. In the transformation event obtained by the application, the inserted exogenous gene is positioned at a non-functional site of a corn genome, the expression of other genes of a receptor plant is not influenced, and the good agronomic characters of the transgenic corn plant are maintained while insect resistance and herbicide tolerance characteristics are obtained.

Description

Insect-resistant herbicide tolerant corn transformation events

Technical Field

The application relates to the technical field of plant biology, in particular to a creation method, a detection method and application of an insect-resistant herbicide-tolerant corn transformation event.

Background

Corn is an important feed and industrial raw crop, which is a crop with the largest planting area in China and has a long-term self-sufficiency, but the import amount is increased year by year since 2010.

The corn borer is commonly called corn borer, and the damage of the corn borer is one of important biological disasters causing perennial yield reduction of the corn, and seriously influences the yield and the quality of the corn, including Asian corn borer (Ostrinia furnacalis) and European corn borer (Ostrinia nubilalis). China is the multiple and repeat region of the asian corn borer (Ostrinia furnacalis) and occurs on a large scale almost every two years. The yield of the corn damaged by the corn borers is reduced by 10-15% in the common year, the yield of the corn in the growing year can be reduced by more than 30%, and even the corn is harvested absolutely. Because of the harm of the corn borers, the corn is lost by 600-900 ten thousand tons every year. The corn borers not only directly cause the loss of the corn yield, but also induce and aggravate the occurrence of the corn ear rot, so that the quality of the corn is reduced. At present, the main mode for preventing and controlling the corn borers is mainly pesticide prevention and control. The use of a large amount of pesticides not only increases the planting cost, but also destroys the ecological environment. The weeds in the field compete with crops for water, fertilizer, light energy and growth space, are intermediate hosts for damaging crop germs and pests, and are one of important biological limiting factors for increasing the yield of crops. The area of crops seriously damaged by weeds all year round in China is up to 12 hundred million acres, wherein 1.9 hundred million acres of corn are obtained. At present, the widely adopted selective herbicide has large application amount and long residual period, and is easy to influence the normal growth of the next-stubble crops. The biocidal herbicides such as glufosinate-ammonium and the like have the characteristics of high efficiency, low toxicity, easy degradation, no residue and the like, but have no selectivity in weeding and cannot be directly used in the growth period of crops. The plant transgenic breeding technology has the advantages of strong purposiveness, short period, high efficiency, capability of realizing the transfer of excellent genes among different species and the like. Since the first commercialization of transgenic crops in 1996, this technology has brought about tremendous changes to global agriculture.

For transgenic corn, cry1Ac, cry1F and other genes for preventing lepidoptera corn borer are taken as main genes, besides, a plurality of lines with coleoptera resistance such as MON88017 and other lines with corn rootworm resistance obtained by using Cry3 genes such as MON863 are put into commercial production.

Worldwide, over 40 transgenic Bt resistant corn varieties have been approved by 26 countries for commercial production or feed and food processing in 1996 to date, such as Monsanto USA MON810 and MON 89034.

Disclosure of Invention

In one aspect, the present application provides a nucleic acid molecule comprising: i) Comprises a sequence shown by nucleotides 381 to 780 and/or nucleotides 10815 to 11214 of SEQ ID NO.1, or a fragment or variant or complementary sequence thereof; ii) comprises the sequence shown at nucleotides 381-780 and 6239-6338 of SEQ ID NO.1, or a fragment or variant or complement thereof; iii) Comprises a sequence shown as nucleotides 6239-6338 and nucleotides 10815-11214 of SEQ ID NO.1, or a fragment or variant thereof or a complementary sequence thereof; or iv) comprises the sequence shown by nucleotides 381 to 780, nucleotides 6239 to 6338 and nucleotides 10815 to 11214 of SEQ ID NO.1, or a fragment or variant or complement thereof.

In one embodiment, the nucleic acid molecule provided herein comprises the sequence shown in SEQ ID No.1, or a fragment or variant thereof or the complement thereof.

In another embodiment, a nucleic acid molecule provided herein comprises the following expression cassette: a first expression cassette for expressing a glufosinate-resistant gene, which has a sequence shown as 748-2288 th nucleotides of SEQ ID NO. 1; a second expression cassette for expressing insect-resistant genes, which has a sequence shown as 2620-6959 nucleotides of SEQ ID NO. 1; and a third expression cassette for expressing glyphosate-resistant gene, such as the sequence shown by nucleotides 6968-10892 of SEQ ID NO. 1.

In another embodiment, the nucleic acid molecule provided herein is obtained by introducing into the genome of maize: a first expression cassette for expressing a glufosinate-ammonium-resistant gene, which has a sequence shown as 748-2288 th nucleotides in SEQ ID NO. 1; a second expression cassette for expressing insect-resistant genes, which has a sequence shown as 2620-6959 th nucleotides of SEQ ID NO. 1; and a third expression cassette for expressing glyphosate-resistant gene, such as the sequence shown by nucleotides 6968-10892 of SEQ ID NO. 1.

The nucleic acid molecules provided herein are present in a maize plant, seed, plant cell, progeny plant or plant part.

In another aspect, the present application provides a probe for detecting a maize transformation event comprising the sequence shown at nucleotides 381-780 or nucleotides 10815-11214 of SEQ ID NO.1, or a fragment thereof or a variant or complement thereof.

Also provided herein are primer pairs for detecting a maize transformation event that are capable of specific amplification to produce a sequence comprising nucleotides 381-780 or nucleotides 10815-11214 of SEQ ID NO.1, or a fragment or variant or complement thereof.

In one embodiment, the primer pair is: i) A primer pair which specifically recognizes a sequence shown by nucleotides 381-780 of SEQ ID NO. 1; ii) a primer pair which specifically recognizes a sequence comprising nucleotides 10815-11214 of SEQ ID NO. 1; iii) A forward primer which specifically recognizes a sequence shown by nucleotides 381 to 780 of SEQ ID NO.1, and a reverse primer which specifically recognizes a sequence shown by nucleotides 681 to 10915 of SEQ ID NO. 1; iv) a forward primer specifically recognizing a sequence consisting of nucleotides 681 to 10915 of SEQ ID NO.1, and a reverse primer specifically recognizing a sequence consisting of nucleotides 10815 to 11214 of SEQ ID NO. 1.

In one embodiment, the primer pair provided herein is the nucleotide sequence shown in SEQ ID No.8 and SEQ ID No.9 or the complementary sequence thereof; or the nucleotide sequences shown in SEQ ID No.10 and SEQ ID No.11 or the complementary sequences thereof.

In addition, kits or microarrays for detecting maize transformation events comprising the probes described above and/or the primer pairs described above are also provided.

In yet another aspect, the present application provides a method for detecting a maize transformation event, comprising detecting the presence or absence of the transformation event in a test sample using: the above-mentioned probe; the above-mentioned primer pair; the above-mentioned probe and primer pair; or a kit or microarray as described above.

The present application also provides a method of breeding maize, the method comprising the steps of: 1) Obtaining maize comprising the nucleic acid molecule described above; 2) Subjecting the corn obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain progeny plants, seeds, plant cells, progeny plants or plant parts; and optionally, 3) carrying out resistance identification of herbicides glufosinate and glyphosate and stem borers and/or armyworms on the progeny plants obtained in the step 2), and detecting whether the transformation event exists in the progeny plants by using the method.

Further, the present application also provides corn plants, seeds, plant cells, progeny plants or plant parts, etc. obtained by the above-described methods, as well as articles made from these corn plants, seeds, plant cells, progeny plants or plant parts, etc., including food, feed or industrial materials, etc.

Further, the present application provides a method of controlling a lepidopteran pest population comprising contacting said lepidopteran pest population with a corn plant, seed, plant cell, progeny plant or plant part obtained by the above-described method.

The present application also provides a method of killing a lepidopteran pest comprising contacting said lepidopteran pest with a pesticidally-effective amount of a corn plant, seed, plant cell, progeny plant or plant part obtained by the above method.

The present application also provides a method of reducing damage to corn by a lepidopteran pest, comprising introducing into the genome of corn: a first expression cassette for expressing a glufosinate-resistant gene, which has a sequence shown as 748-2288 th nucleotides of SEQ ID NO. 1; a second expression cassette for expressing insect-resistant genes, which has a sequence shown as 2620-6959 nucleotides of SEQ ID NO. 1; and a third expression cassette for expressing glyphosate-resistant gene, such as the sequence shown by nucleotides 6968-10892 of SEQ ID NO. 1.

In particular embodiments, the lepidopteran pest described in the above methods is asian corn borer (Ostrinia furnacalis), european corn borer (Ostrinia nubilalis), or oriental armyworm (Mythimna separate (Walker)).

Drawings

Fig. 1 is a schematic structural diagram of vector pzhzhzhh 35006, in which:

ubiquitin promoter from maize

Omega sequence derived from tobacco mosaic virus gene expression enhancing elements

Kozak sequence in eukaryotic mRNA sequence for translation initiation

cry1Ab/cry1Ac Zm optimized Bt gene sequence

PolyA polyadenylation sequence

nos terminator of Agrobacterium nopaline synthase gene

T-Border (right) T-DNA right Border sequence

CaMV 35S promoter cauliflower mosaic virus 35S promoter

Bar anti-glufosinate gene sequence

CaMV 35S terminator for cauliflower mosaic virus

T-Border (left) T-DNA left Border sequence

Kanamycin (R) Kanamycin resistance sequence

pBR322 ori pBR322 initiation region sequence

pBR322 bom pBR322 framework region sequence

pVS1 rep pVS1 replicon

pVS1 sta pVS1 transcriptional initiation region

Glyphosate-resistant gene sequence of cp4Zm coding EPSPS protein

Omega sequence1 derived from tobacco etch virus gene expression enhancing element

ubiquitin4promoter from sugarcane

bp base pair

FIG. 2 is a photograph of the plants after 4-5 days of 250 ml/acre of glufosinate herbicide spraying, wherein:

a is ZZM030, the plant grows normally, and no damage symptom exists;

b is non-transgenic negative control wild type 249, and has dry, green, spot and growth retardation of leaf and obvious phytotoxicity symptoms.

FIG. 3 is a photograph of a plant one week after 200 ml/acre of glyphosate herbicide "farmlands" was sprayed, wherein:

a is ZZM030, the plant grows normally, and no damage symptom exists;

b is a non-transgenic negative control wild type Zhanxiang 249, which has dry leaves, chlorosis, growth retardation and obvious phytotoxicity symptoms.

FIG. 4 is a photograph of a field experiment for identifying resistance of plant borers, white spots in the photograph are wormholes, wherein:

a is ZZM030 leaf, pinhole-shaped wormhole, which is rare and scattered;

b is non-transgenic negative control wild type Xiang 249 leaves, mung bean big and small wormholes, individually present as short strips Kong Huashe.

FIG. 5 is a photograph of a field experiment for identifying the armyworm resistance of a plant, wherein the edge of a leaf and the missing part on the leaf are wormholes eaten by armyworms, and the following steps are included:

a is ZZM blade;

b is non-transgenic negative control wild type leaf of 249.

FIG. 6 is a ZZM030Southern blot copy number assay result, wherein:

FIG. 6A is the result of copy number detection of the inserted cry1Ab/cry1Ac ZM gene, wherein: lane 1, dna molecular markers; lane 2, blank; lane 3, hindIII digested ZZM genomic DNA hybridized to cry1Ab/cry1Ac ZM specific probe; lane 4, hindIII digested wild-type 249 genomic DNA hybridized to cry1Ab/cry1Ac ZM specific probe as negative control; lane 5, kpnI digested ZZM genomic DNA hybridized to cry1Ab/cry1Ac ZM specific probe; lane 6, kpnI digested wild-type 249 genomic DNA hybridized with cry1Ab/cry1Ac ZM specific probe as negative control; lane 7, ecoRI digested ZZM030 genomic DNA hybridized to cry1Ab/cry1Ac ZM specific probe as positive control; lane 8, ecoRI digested wild-type 249 genomic DNA hybridized to cry1Ab/cry1Ac ZM specific probe as negative control; lane 9, ecoRI digested plasmid and wild type 249 genomic DNA hybridized to cry1Ab/cry1Ac ZM specific probe as positive control;

FIG. 6B shows the bar gene insert copy number assay results, wherein: lane 1, dna molecular markers; lane 2, blank; lane 3, hindIII digested ZZM030 genomic DNA hybridized to a bar specific probe; lane 4, hybridization of HindIII digested wild-type 249 genomic DNA to a bar specific probe as a negative control; lane 5, ecoRI-digested ZZM030 genomic DNA hybridized to bar specific probe; lane 6, ecoRI-digested wild-type 249 genomic DNA hybridized to bar-specific probe as negative control; lane 7, kpnI digested ZZM030 genomic DNA hybridized to bar specific probe as positive control; lane 8, kpnI digested wild-type 249 genomic DNA hybridized with bar-specific probe as negative control; lane 9, hybridization of the cleaved plasmid from KpnI and the genomic DNA of wild-type 249 to a bar-specific probe, as a positive control;

FIG. 6C shows the cp4Zm gene insert copy number assay results, wherein: lane 1, dna molecular markers; lane 2, blank; lane 3, hindIII digested ZZM030 genomic DNA hybridized to cp4Zm specific probe; lane 4, hybridization of HindIII-digested wild-type 249 genomic DNA with cp4 Zm-specific probe, as a negative control; lane 5, kpnI digested ZZM030 genomic DNA hybridized to cp4Zm specific probe; lane 6, hybridization of KpnI digested wild-type 249 genomic DNA with cp4Zm specific probe as a negative control; lane 7, ecoRI-digested ZZM030 genomic DNA hybridized to cp4Zm specific probe as a positive control; lane 8, ecoRI-digested wild-type 249 genomic DNA hybridized with cp4 Zm-specific probe as negative control; lane 9, ecoRI-digested plasmid and wild-type 249 genomic DNA hybridized to the cp4 Zm-specific probe as a positive control.

FIG. 7 is the result of ZZM030 event specific PCR assay, wherein:

FIG. 7A shows the left border detection result;

FIG. 7B shows the right boundary detection result;

wherein lanes 1-4 are: sterile water, genomic DNA of Xiang 249, genomic DNA of ZZM030, and genomic DNA of non-ZZM event obtained after transformation with the same vector.

Detailed Description

The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise indicated, terms are to be understood in accordance with their ordinary usage by those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards and other publications, etc., cited herein are incorporated by reference in their entirety.

As used herein, "maize" is any maize plant and includes all plant varieties that can be bred with maize, including whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, and plant cells intact in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like.

It is well known to those skilled in the art that expression of foreign genes in plants has a positional effect, i.e., is influenced by the location of insertion into the chromosome, which may be due to a chromosomal structure or transcriptional regulatory elements near the integration site. Therefore, it is usually necessary to produce hundreds of different transformation events and to screen out excellent transformation events with desired exogenous gene expression levels and patterns for commercial production applications.

An excellent transformation event can be achieved by transferring exogenous genes into germplasm of other genetic backgrounds by means of conventional breeding methods, i.e., sexual crosses, whose progeny retain the transgene expression characteristics of the original transformants. The present application relates to superior conversion events ZZM030 by screening out from a number of conversion events.

In this application, "transformation event ZZM030" refers to a maize plant that has been genetically transformed by agrobacterium mediated transformation with maize inbred line 249 as the recipient to obtain an exogenous gene insert (T-DNA insert) inserted at a specific genomic site, wherein the exogenous gene insert comprises the following three genes: insect-resistant gene, glufosinate-resistant gene and glyphosate-resistant gene. According to the transformation event ZZM030 obtained by the application, the inserted exogenous gene is located at a non-functional site of a corn genome, the expression of other genes of a receptor plant is not influenced, and the good agronomic characters of the transgenic corn plant are maintained while the insect-resistant and herbicide-resistant characteristics of the transgenic corn plant are obtained.

In a specific example, the T-DNA insert obtained after the transgene has the sequence shown by nucleotides 681-10915 of SEQ ID NO. 1. Transformation event ZZM030 may refer to this transgenic process, may also refer to a T-DNA insert within the genome resulting from this process, or a combination of a T-DNA insert and flanking sequences, or may refer to a maize plant resulting from this transgenic process. Transformation event ZZM030 may also refer to progeny plants resulting from vegetative propagation, sexual propagation, doubling or doubling of the above plants, or a combination thereof.

In other embodiments, the event is also applicable to plants obtained by transforming other plant recipient varieties with the same foreign gene (shown as nucleotide 681-10915 of SEQ ID NO: 1) and inserting the T-DNA insert into the same genomic position. Suitable plants include monocotyledonous plants such as rice, wheat, oats, barley, highland barley, millet, sorghum, sugarcane, and the like.

In the present application, a T-DNA insert (nucleotides 681-10915) was obtained with nucleotides 1-680 of SEQ ID NO:1 as the left flank and nucleotides 10916-11375 of SEQ ID NO:1 as the right flank. The flanking sequences are not limited to nucleotides 1-680 and 10916-11375 of SEQ ID NO.1, because the flanking sequences are listed only to indicate the position of the T-DNA insert in the genome, i.e., the insertion point to the left of the T-DNA insert is located on chromosome 4, 40636901bp; the right insertion point of the T-DNA insert is located on chromosome 4, 40636883bp. Thus, the flanking sequences of the present application may be extended bilaterally according to the genomic sequence, i.e., the left flanking sequence may be extended downstream of 40636901bp of chromosome 4 and the right flanking sequence may be extended upstream of 40636883bp of chromosome 4.

Since transformation event ZZM030 produces a T-DNA insert that is inserted into a specific site in the genome, its insertion site is specific and can be used to detect whether transformation event ZZM030 is contained in a biological sample. In particular embodiments, any sequence comprising the site of junction of a T-DNA insert with a flanking sequence of transformation event ZZM030 can be used to detect transformation event ZZM030 of the present application, including, but not limited to, one or more of the following sequences comprising an upstream insertion site (the site of junction of the left flank sequence with the T-DNA insert) or a downstream insertion site (the site of junction of the right flank sequence with the T-DNA insert), or a fragment thereof, or a variant thereof, or a complement thereof: i) Comprises a sequence shown as nucleotides 381-780 of SEQ ID NO. 1; ii) comprises the sequence shown as nucleotides 1 to 898 of SEQ ID NO. 1; iii) Comprises a sequence shown as nucleotides 6239-6338 of SEQ ID NO. 1; iv) comprises the sequence as shown in nucleotides 10815-11214 of SEQ ID NO. 1; v) comprises the sequence shown as nucleotides 10578-11373 of SEQ ID NO. 1; vi) comprises the sequence shown as nucleotides 381-11241 of SEQ ID NO. 1; vii) comprises the sequence shown in SEQ ID NO. 1.

In specific examples, sequences useful for detecting transformation event ZZM030 herein are sequences comprising an upstream insertion site or fragments or variants thereof or complements thereof, such as the sequences shown as nucleotides 381-780 of SEQ ID No.1 or the sequences shown as nucleotides 1-898 of SEQ ID No.1, or sequences comprising a downstream insertion site, e.g., the sequences shown as nucleotides 10815-11214 of SEQ ID No.1 or the sequences shown as nucleotides 10578-11373 of SEQ ID No.1, or a combination of a sequence comprising an upstream insertion site and a sequence comprising a downstream insertion site.

In another example, a sequence useful for detecting a transformation event ZZM030 herein is a combination of a sequence comprising an upstream insertion site or a fragment thereof or a variant thereof or a complement thereof and a sequence comprising a T-DNA insert or a fragment thereof or a variant thereof or a complement thereof, e.g., a sequence comprising nucleotides 381 to 780 of SEQ ID NO.1 or a sequence comprising nucleotides 1 to 898 of SEQ ID NO.1, a combination comprising a sequence comprising nucleotides 6239 to 6338 of SEQ ID NO.1 or a sequence comprising nucleotides 681 to 10915 of SEQ ID NO. 1.

In another example, a sequence that can be used to detect a transformation event ZZM030 of the present application is a combination of a sequence comprising a downstream insertion site or a fragment thereof or a variant thereof or a complement thereof and a sequence comprising a T-DNA insert or a fragment thereof or a variant thereof or a complement thereof, e.g., a sequence comprising nucleotides 10815-11214 of SEQ ID No.1 or a sequence comprising nucleotides 10578-11373 of SEQ ID No.1, and a sequence comprising nucleotides 6239-6338 of SEQ ID No.1 or a sequence comprising nucleotides 681-10915 of SEQ ID No. 1.

In another example, a sequence that can be used to detect transformation event ZZM030 herein is a sequence comprising nucleotides 381 to 11241 of SEQ ID NO:1 or a fragment or variant or complement thereof, or a sequence comprising SEQ ID NO:1 or a fragment or variant or complement thereof.

Thus, primer pairs, probes, and combinations of primer pairs and probes that are capable of specifically detecting the site of junction of the T-DNA insert of transformation event ZZM030 with flanking sequences can all be used to detect transformation event ZZM of the present application.

As used herein, "nucleotide sequence" includes reference to deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, nucleotide sequences are written from left to right in the 5 'to 3' direction.

In some embodiments, the present application also relates to fragments of nucleic acid sequences, which refer to portions of smaller fragments that are incomplete in a complete portion. For example, SEQ ID NO:1 comprises SEQ ID NO:1, at least about 10 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, or at least about 50 nucleotides of the complete sequence or more.

In some embodiments, changes may be made to the nucleic acid sequences of the present application to make conservative amino acid substitutions. In certain embodiments, substitutions that do not alter the amino acid sequence of the nucleotide sequences of the present application can be made according to monocot codon preferences, e.g., codons encoding the same amino acid sequence can be substituted with monocot preferred codons without altering the amino acid sequence encoded by the nucleotide sequence. In some embodiments, the present application also relates to variants of the nucleic acid sequences. Generally, variants of a particular nucleic acid fragment will have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or more sequence identity, or the complement thereof, to the particular nucleotide sequence. Such variant sequences include additions, deletions or substitutions of one or more nucleic acid residues, which may result in the addition, removal or substitution of the corresponding amino acid residue. Sequence identity is determined by sequence alignment programs known in the art, including hybridization techniques. Nucleotide sequence variants of the embodiments may differ from the sequences of the present application by as little as 1-15 nucleotides, as little as 1-10 (e.g., 6-10), as little as 5, as little as 4, 3, 2, or even 1 nucleotide.

As used herein, a "probe" is an isolated polynucleotide, complementary to a strand of a target polynucleotide, to which is attached a conventional detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent or enzyme.

In a specific embodiment, a DNA probe for detecting transformation event ZZM030 as provided herein, comprises a DNA comprising SEQ ID NO:1 or a sequence that is fully complementary thereto, which DNA probe hybridizes under stringent hybridization conditions to a nucleotide sequence comprising an upstream insertion site or a downstream insertion site and does not hybridize under stringent hybridization conditions to a nucleotide sequence not comprising an upstream insertion site or a downstream insertion site.

In a specific example, the probe provided herein comprises the sequence shown by nucleotides 381 to 780 or nucleotides 10815 to 11214 of SEQ ID NO.1, or a fragment or variant or complement thereof. As used herein, a "primer" is an isolated polynucleotide that anneals to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then stretches along the target DNA strand by means of, for example, a DNA polymerase. Primer pairs are directed to their target polynucleotide amplification use, for example, by Polymerase Chain Reaction (PCR) or other conventional nucleic acid amplification methods.

In particular embodiments, the primer pair for detecting transformation event ZZM030 provided herein comprises a first DNA molecule and a second DNA molecule different from the first DNA molecule, wherein the first and second DNA molecules each comprise SEQ ID NO:1 or the complete complement thereof, and wherein the first DNA molecule is present in SEQ ID NO:1 and the second DNA molecule are present in SEQ ID NO:1 when used in conjunction with DNA from transformation event ZZM030 in an amplification reaction, the DNA primers produce an amplicon for detection of transformation event ZZM030DNA in a sample, and wherein the amplicon comprises the sequence shown at nucleotides 381-780 or nucleotides 10815-11214 of SEQ ID NO:1, or a fragment or variant or complement thereof.

In a specific embodiment, the primer pair provided herein is a primer pair that specifically recognizes a sequence comprising nucleotides 381 to 780 or 1 to 898 of SEQ ID NO. 1.

In a specific embodiment, the primer pair provided herein is a primer pair that specifically recognizes a sequence comprising nucleotides 10815-11214 or 10578-11373 of SEQ ID NO. 1.

In specific embodiments, the primer pairs provided herein are: i) A primer pair which specifically recognizes a sequence shown by nucleotides 381 to 780 or 1 to 898 of SEQ ID NO. 1; and ii) a primer pair which specifically recognizes a sequence comprising the sequence shown by nucleotides 10815-11214 or 10578-11373 of SEQ ID NO. 1; alternatively, the primer pair comprises: a forward primer specifically recognizing a sequence represented by nucleotides 381 to 780 or 1 to 898 of SEQ ID NO.1, and a reverse primer specifically recognizing a sequence represented by nucleotides 10815 to 11214 or 10578 to 11373 of SEQ ID NO. 1.

In another embodiment, the primer pairs provided herein are: i) A primer pair which specifically recognizes a sequence shown by nucleotides 381 to 780 or 1 to 898 of SEQ ID NO. 1; and ii) a primer pair which specifically recognizes a sequence shown by nucleotides 6239 to 6338 or 681 to 10915 of SEQ ID NO. 1; alternatively, the primer pair comprises: a forward primer specifically recognizing a sequence shown by nucleotides 381 to 780 or 1 to 898 of SEQ ID NO.1, and a reverse primer specifically recognizing a sequence shown by nucleotides 6239 to 6338 or 681 to 10915 of SEQ ID NO. 1.

In another embodiment, the primer pairs provided herein are: i) A primer pair which specifically recognizes a sequence shown by the 6239-6338 th or 681-10915 th nucleotides of SEQ ID NO. 1; and ii) a primer pair which specifically recognizes a sequence comprising the sequence shown by nucleotides 10815-11214 or 10578-11373 of SEQ ID NO. 1; alternatively, the primer pair comprises: a forward primer which specifically recognizes a sequence shown by the 6239-6338 th or 681-10915 th nucleotides of SEQ ID NO.1, and a reverse primer which specifically recognizes a sequence shown by the 10815-11214 th or 10578-11373 th nucleotides of SEQ ID NO. 1.

In another embodiment, the primer pairs provided herein are: i) A primer pair which specifically recognizes a sequence shown by nucleotides 381 to 780 or 1 to 898 of SEQ ID NO.1, ii) a primer pair which specifically recognizes a sequence shown by nucleotides 6239 to 6338 or 681 to 10915 of SEQ ID NO.1, and iii) a primer pair which specifically recognizes a sequence shown by nucleotides 10815 to 11214 or 10578 to 11373 of SEQ ID NO. 1.

In another embodiment, the primer pair provided herein is a primer pair that specifically recognizes a sequence comprising SEQ ID NO. 1.

In a specific example, the primer pair is the nucleotide sequence shown as SEQ ID No.8 and SEQ ID No.9 or the complementary sequence thereof; or the nucleotide sequences shown in SEQ ID No.10 and SEQ ID No.11 or the complementary sequences thereof.

Methods for designing and using primers and probes are well known in the art and are described, for example, in Sambrook et al, A handbook of Molecular cloning experiments (Sambrook J & Russell DW, molecular cloning: a laboratory Manual, 2001) and in the Molecular Biology laboratory Manual (Current Protocols in Molecular Biology) published by Wiley-Blackwell.

As used herein, "kit" or "microarray" refers to a set of reagents or chips for the purpose of identification and/or detection of corn transformation event ZZM030 in a biological sample. For the purpose of quality control (e.g., purity of seed lot), detection of event ZZM030 in or comprising plant material or material derived from plant material, such as, but not limited to, food or feed products, kits or chips can be used, and components thereof can be specifically adjusted.

In particular embodiments, a kit or probe provided herein includes any one of the probes or any one of the primer pairs provided herein. In another specific embodiment, a kit or probe provided herein comprises any one of the probes provided herein or a combination of any one of the primer pairs.

In addition, transgenic corn plants, progeny, seeds, plant cells or plant parts and preparations thereof, including but not limited to food, feed or industrial materials, are provided. The plants, progeny, seeds, plant cells, plant parts, and preparations thereof all comprise a nucleic acid molecule sequence that is detectable as a site of engagement of the T-DNA insert provided herein with the flanking sequences.

Further, the present application also provides a method of breeding maize comprising the steps of: 1) Obtaining maize comprising a nucleic acid molecule sequence comprising the junction site of the T-DNA insert provided herein with flanking sequences; 2) Subjecting the corn obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain progeny plants, seeds, plant cells, progeny plants or plant parts; and optionally step 3), subjecting the corn plants obtained in step 2) to herbicide-resistant glufosinate-ammonium and glyphosate and insect-resistant identification, and detecting the presence or absence of transformation event ZZM030 therein using probes, primer pairs, kits or arrays provided herein.

In addition, the present application provides methods of controlling weeds in a field, as well as methods of controlling or killing lepidopteran pests.

In a specific embodiment, a method of controlling weeds in a field provided herein comprises planting corn plants comprising transformation event ZZM in a field and applying an effective amount of glyphosate and glufosinate herbicide in the field that is capable of controlling weeds without damaging the transgenic corn plants comprising event ZZM030.

In particular embodiments, methods of controlling or killing a lepidopteran pest provided herein comprise contacting the lepidopteran pest with an effective amount of a corn plant of transformation event ZZM, or feeding the lepidopteran pest with an effective amount of a corn plant of transformation event ZZM030, or feeding the lepidopteran pest with an effective amount of a corn plant of transformation event ZZM030. The lepidopteran pests include, but are not limited to, asian corn borer (Ostrinia furnacalis), european corn borer (Ostrinia nubilalis), oriental armyworm (Mythimna separate (Walker)), and the like.

As used herein, "effective amount" or "pesticidally effective amount" refers to the amount of a substance or organism having pesticidal activity that is present in the pest environment.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and substance of the invention and are intended to be included within the scope of the present application.

Unless otherwise indicated, the examples follow conventional experimental conditions, such as Sambrook et al, handbook of Molecular cloning experiments (Sambrook J & Russell DW, molecular cloning: a laboratory Manual, 2001), or following the conditions suggested by the manufacturer's instructions.

Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

The maize variety materials related to the following examples are all provided by the Chinese seed group company ltd, wherein the maize inbred line 249 is the female parent of the great wall 799 maize variety, maize germplasm resources introduced abroad are used as materials, and the maize inbred line materials are obtained by inbreeding separation and strict selection through a pedigree method and breeding in 1996 after 10 generations.

Example 1 vector construction

1. Synthesis of insect-resistant Gene

The insect-resistant gene cry1Ab/cry1Ac ZM disclosed in PCT International application WO2017012577A1 by the present applicant, which is expressed in plants and produces an insect-resistant effect, is utilized. The gene is based on the N-terminal 608 amino acid sequences of Cry1Ab and Cry1Ac which are fused and modified, the coding sequence is replaced by using codons preferred by plants, and AT-rich sequences which cause unstable plant transcription and common restriction enzyme cutting sites existing in a DNA sequence are corrected and eliminated by a method of replacing the codons. Meanwhile, a section of omega sequence with 67 nucleotides and Kozak sequence with 3 nucleotides (ACC) are added at the 5' end to enhance the translation efficiency of eukaryotic genes. A135 bp polyA sequence was added to the 3' end. The gene encodes a protein containing 3 functional segments, wherein two functional regions at the N end are highly homologous with corresponding parts of Cry1Ab, and a functional region at the C end is highly homologous with Cry1Ac. An insect-resistant gene cry1Ab/cry1Ac Zm shown by bases 4624 th to 6670 th of SEQ ID NO.1 is synthesized by an artificial synthesis mode.

2. Synthesis of herbicide-resistant gene exogenous gene

The codon preference optimization of the glyphosate gene cp4 coding region sequence is carried out by utilizing Vector NTI software, an expression enhancing element omega sequence1 is added at the 5' end, and the modified DNA sequence is named cp4Zm. Synthesizing the glyphosate herbicide resistant gene shown as 8954 th to 10611 th bases of SEQ ID NO.1 in an artificial synthesis way.

The sequence of the bar Gene refers to the sequence shown in the Gene bank accession number X17220.1, and the glufosinate-ammonium-resistant herbicide Gene shown as the base groups 957 to 1508 of the SEQ ID NO.1 is synthesized in an artificial synthesis mode.

3. Vector construction

HindIII and PstI cleavage sites are added to the 5 'end and PmeI cleavage sites are added to the 3' end of the synthesized cry1Ab/cry1Ac Zm, and the synthesized sequence is cloned into a Puc57simple vector, which is named pZZ01194.

The intermediate vector pZZ00005 containing ubiquitin promoter (5 '-terminal having HindIII cleavage site and 3' -terminal having BamHI cleavage site) was digested simultaneously with restriction enzymes HindIII and BamHI, and the resulting sticky ends were filled in with T4DNA polymerase to obtain ubiquitin promoter fragment.

The vector containing the ubiquitin promoter-cry1Ab/cry1Ac Zm fragment was obtained by treating pZZ01194 with restriction enzyme PstI, filling in the resulting sticky ends with T4DNA polymerase, and ligating ubiquitin promoter by blunt end ligation, and was named pZZ01201.

The nos terminator sequence was obtained by single-digesting the existing intermediate vector pZZ01188 containing nos terminator (EcoRI cleavage site at 5 'end and PmeI, ecoRI site at 3' end) with restriction enzyme EcoRI, and filling in the resulting sticky ends with T4DNA polymerase.

PmeI treated pZZ01201, and nos terminator were ligated together by blunt end ligation to obtain a vector containing ubiquitin promoter-cry1Ab/cry1Ac Zm-nos terminator fragment, which was named pZZ01205.

Intermediate vector pZZ00015 (containing CaMV 35S promoter-bar-CaMV 35S terminator and ubiquitin promoter-egfp-nos terminator expression elements) was subjected to restriction enzyme HindIII and PmeI to remove ubiquitin promoter-egfp-nos terminator. The pZZ01205 vector was treated with restriction enzymes HindIII and PmeI to obtain the ubiquitin promoter-cry1Ab/cry1Ac Zm-nos terminator fragment. The two were ligated to obtain an expression vector containing two expression cassettes of ubiquitin promoter-cry1Ab/cry1Ac Zm-nos terminator and CaMV 35S promoter-bar-CaMV 35S terminator, which was designated as pZHZHZH 25017.

The intermediate vector pZZ01337 (CaMV 35S promoter-cp4Zm-nos terminator) was excised with restriction enzymes HindIII and BamHI. The intermediate vector pZZ00033 was used for the restriction enzymes HindIII and BamHI to obtain ubiquitin4promoter. The two were ligated to obtain a vector containing the ubiquitin4 promoter-cp4Zm-nos terminator fragment, which was named pZZ01383.

The pZZ01383 was double digested with restriction enzymes HindII and PmeI to obtain the ubiquitin4 promoter-cp4Zm-nos terminator fragment, and the resulting sticky ends were filled in with T4DNA polymerase.

The plant expression vector containing three expression cassettes of ubiquitin promoter-cry1Ab/cry1Ac Zm-nos terminator, caMV 35S promoter-bar-CaMV 35S terminator and ubiquitin4 promoter-4 Zm-nos terminator was obtained by treating pZHZHZH 25017 with restriction enzyme PmeI and ligating-in ubiquitin4 promoter-cp4Zm-nos terminator by blunt-end ligation, and named as pZHZHZH 35006, and its physical map is shown in FIG. 1.

Example 2 obtaining of transgenic maize

Transgenic maize is obtained using agrobacterium-mediated genetic transformation methods.

The plasmid DNA of the vector pZHZHZH 35006 was transformed into Agrobacterium EHA105 by electric shock method, and identified for use.

The efficient transgenic method of the corn backbone inbred line disclosed in the Chinese invention patent application CN104745622A by the applicant is utilized for transformation.

Specifically, young embryos with the length of about 1.5mm are taken for transformation after selfing of the maize inbred line 249. Collecting young embryos of about 200 clusters as a batch, placing the batch in an EP tube, sucking out the suspension, adding an agrobacterium liquid containing 200 mu M acetosyringone, co-culturing for 5min, transferring the young embryos to a co-culture medium, and culturing in the dark for 3 days. Placing the dark-cultured immature embryo on a callus induction culture medium, placing the culture medium on a screening culture medium containing 5mg/L bialaphos after the callus grows out, screening and culturing, and subculturing once every two weeks. When resistant callus grows out, selecting out embryogenic callus with good state, transferring to differentiation culture medium under the culture condition of 26 deg.C, 3000Lux light intensity every day, and illuminating for 16h, wherein two weeks later, regeneration plantlet appears. Transferring the regenerated plantlets into a rooting culture medium, and transplanting the plantlets into a small pot mixed with nutrient soil and vermiculite (1: 3) after secondary roots grow out of the plantlets.

The obtained transformed seedlings are subjected to transgenic positive detection according to the following steps, and transgenic positive plants are selected.

(1) DNA extraction

The corn genomic DNA was extracted using DNAsecure Plant Kit novel Plant genomic DNA extraction Kit (centrifugal column type) from Tiangen Biochemical technology.

(2)PCR

The following reagents were thawed from a-20 ℃ freezer: 10-fold PCR buffer (Takara), deoxynucleotide mix (10mM, sigma), forward primer including SEQ ID NO:2 (CSP 759): 5'-CACGCAGATTCCAGCGGTCAA-3'; reverse primer SEQ ID NO:3 (CSP 760): 5'-GACGAGGTGAAGGCGTTAGCA-3') and maize leaf DNA template. After thawing all reagents, centrifuge for several seconds and place on ice for use. And preparing a mixed solution of a PCR reaction system, uniformly mixing, and centrifuging for several seconds. PCR reaction (20. Mu.L): mu.L of 10-fold PCR buffer (Takara), 0.5. Mu.L of deoxynucleotide mixture (10mM, sigma), 0.8. Mu.L of Forward and reverse primersPrimer mix (5. Mu.M), 0.2. Mu. L r-Taq (5U, takara), the remainder dd H ₂ And O. And subpackaging the mixed solution into 200 mu L PCR tubes, adding 1 mu L corn leaf DNA template, and marking different samples for distinguishing. And (3) putting the PCR reaction tube into an ABI 9700 type PCR amplification instrument, selecting a preset PCR amplification program, and starting to operate the reaction. The PCR reaction program is: pre-denaturation at 94 ℃ for 2min;30 cycles: denaturation at 94 ℃ 30sec, annealing at 58 ℃ 30sec, extension at 72 ℃ for 30sec; finally, extension is carried out for 5min at 72 ℃.

(3) Agarose gel electrophoresis detection

After the PCR is finished, 5 μ L of PCR product is taken for agarose gel electrophoresis detection. A1.5% agarose gel was prepared, electrophoresed at 150V for 25min, stained in Ethidium Bromide (EB) for 10min, and photographed in an ultraviolet gel imaging system.

(4) Determination of the result

The material capable of amplifying the 333bp band is a transgenic positive plant, and the material incapable of amplifying the band is a transgenic negative plant.

Transplanting the transgenic positive plant into a large flowerpot to obtain T ₀ And (5) plant generation.

Example 3 resistance identification of transgenic maize

T ₀ Selfing the plant, the obtained seed is T ₁ And (5) seed generation. Will T ₁ Seeding the seeds in a greenhouse to obtain T ₁ And (5) plant generation. Repeating the above process until T is obtained ₄ And (5) seed generation.

For T ₁ To T ₃ And carrying out transgenic positive detection, herbicide tolerance analysis, insect resistance identification and agronomic character analysis on the generation plants. Selecting out plants with positive transgenosis, insect resistance, herbicide tolerance and excellent agronomic character from each generation of plants, and screening the plants in the next generation.

1. Positive detection

The detection method and procedure were as described in example 2.

2. Herbicide tolerance character identification

And (3) sowing the selfed seeds of the positive plants detected in the step (1) into a greenhouse, carrying out herbicide resistance identification on plants in a 6-8 leaf stage, and removing the plants which do not tolerate the herbicide.

(1) Glufosinate herbicide tolerance identification

The glufosinate herbicide 'Baozida' (Basta) used for spraying is produced by Bayer crop science (China) limited company, and the active ingredient is 18% glufosinate-ammonium soluble agent. The recommended dosage of the herbicide is 200-300 ml/mu, and the herbicide is sprayed by adopting the dosage of 250 ml/mu in the recommended concentration. Herbicide tolerance performance was observed and recorded after 4-5 days. The corn plants with the tolerance to glufosinate-ammonium grow normally and have no damage symptom; maize plants sensitive to glufosinate exhibit significant phytotoxicity symptoms including growth arrest, chlorosis, blight, deformity, and the like, until the entire plant dies.

At T ₁ 、T ₂ And T ₃ In the generation plant population, the Chi-square test was performed according to the following formula, based on the actual segregation ratio for which glufosinate resistance was observed and the expected segregation ratio calculated according to mendelian's law of inheritance (table 1): chi shape ² ＝Σ[(|o–e|–0.5) ² /e](ii) a Wherein "o" is an observed value of the number of positive strains or a number of negative strains, "e" is an expected value of the number of positive strains or the number of negative strains, and "0.5" is a Yates analysis correction factor with a degree of freedom of 1.

TABLE 1 transformant ZZM030 expected separation ratio for each generation

* : in the case of single site single copy insertion, the expected segregation ratio calculated according to Mendelian's Law of inheritance.

TABLE 2 transformant ZZM030 Segregation analysis- χ for each generation ² Examination of

In table 2, "observed values" are the actual number of positive plants and the actual number of negative plants observed after glufosinate-ammonium spraying; "expectation value" is according to Mendelian's Law of inheritanceCalculating the theoretical number of transgenic positive plants and the theoretical number of transgenic negative plants according to the table 1; 'chi' for treating rheumatism ² "is a Chi square value calculated according to a Chi square test formula; 'X' type ² _0.05,1 "is at significance level α =0.05, degree of freedom is 1, chachi ² Values obtained from the boundary value table; "probability" is χ ² And chi ² _0.05,1 The result of comparison between the two is when x ² <χ ² _0.05,1 When it is, then P>0.05, indicating no significant difference between observed and expected values. χ in Table 2 ² Test analysis showed that at T ₁ -T ₃ No significant difference (P) was observed between the observed and expected genetic segregation ratios in the investigated generations>0.05 ZZM030 stably inherit between generations according to Mendelian inheritance rule and at T ₃ The generation is homozygous.

(2) Glyphosate herbicide tolerance identification

The glyphosate herbicide "nongda" (Roundup) used for spraying is produced by Monsanto company, and the active ingredient is 41% isopropylamine salt. The recommended dosage of the herbicide in the corn field is 150-250 ml/mu, the herbicide is sprayed by adopting the dosage of 200 ml/mu in the recommended concentration, and the herbicide tolerance performance is observed and recorded after one week. The corn plants with the glyphosate tolerance grow normally without any damage symptom; corn plants sensitive to glyphosate exhibit significant phytotoxicity symptoms including growth inhibition, chlorosis, blight, deformity, and the like, until the entire plant dies.

3. Transgenic corn plant stem borer resistance identification

The stem borer resistance of the plants is identified in the field by adopting a heart-leaf period living body inoculation method.

Inoculating the corn plants when the corn plants grow and develop to the middle heart-leaf stage (7-leaf stage). The insect to be tested was Asiatic corn borer (Ostrinia furnacalis), and about 60 eggs in the black head stage were placed in a centrifuge tube and the tube mouth was closed with a tampon. Placing the centrifuge tube into an incubator with 28 deg.C and 80% humidity, or placing at room temperature, covering with a wet towel, incubating, removing absorbent cotton, and placing into the cardiac plexus. Each plant is inoculated with 10-20 insects. Investigating the damage degree of the heart and leaves of the plants one by one after 2-3 weeks of insect inoculation, and dividing damage grades according to the size and the number of insect holes on damaged leaves, wherein the damage grades are called leaf eating grades. The present application adopted the 9-grade classification criteria established by the International Cooperation group of corn borer (Table 1). The leaf feeding grade was investigated on a plant-by-plant basis, the average value of each plant was taken as the leaf feeding grade of the line for identification, and the anti-borer grade was determined according to the evaluation criteria of table 3.

TABLE 3 evaluation criteria for corn borer resistance field identification

Note: * Tunnel 2.5cm was 1 hole for stem borer evaluation.

* HR: high resistance; r: resisting; MR: resisting; s: feeling; HS: feeling of height

4. Corn transformation event ZZM030

Through the above process, a corn transformation event ZZM030 is finally screened.

FIG. 2 is a photograph of a plant after 4-5 days of 250 ml/mu glufosinate-ammonium herbicide application, wherein in FIG. 2A, ZZM030 shows that the plant grows normally without any damage symptom, and in FIG. 2B, the plant shows that the wild type control 249 shows that the leaves are dry, green, blotchy and growth-arrested, and obvious phytotoxicity symptoms are shown. Indicating that the event exhibits high resistance to glufosinate herbicides.

FIG. 3 is a photograph of a plant after one week of "reach" spraying glyphosate herbicide at 200 ml/acre, wherein FIG. 3A is ZZM030, the plant grows normally without any symptoms of damage. FIG. 3B shows the control 249 of wild type, dry leaves, chlorosis, growth retardation, and marked symptoms of phytotoxicity. Indicating that the event exhibits high resistance to glyphosate herbicide.

FIG. 4 is a photograph of field experiment for identification of plant stem borer resistance, which is carried out by inoculating Asian corn borer in heart-leaf stage living body, wherein white spots are wormholes. FIG. 4A shows ZZM030 leaves with pinhole-like wormholes, which are rare and scattered. FIG. 4B shows non-transgenic negative control leaf 249, mung bean-sized worm holes, individually in short strips Kong Huashe. The event is shown to reach a high resistance level to Asian corn borers, and the average leaf feeding level of the leaves of the event reaches 1.2 to 1.5 (see Table 4).

TABLE 4 determination of the leaf feeding level of the transformed borer in different generations

Remarking: the number of investigated plants n =10; "+" indicates significant difference compared to negative control

Example 4 armyworm resistance identification of maize transformation event ZZM030

1) In the mythimna separata resistance in vitro bioassay experiment I, a first-arrival commercial transgenic corn variety Bt11 is taken as a positive control, the material contains Cry1Ab genes, and the reported material has mythimna separata resistance; receptor material 249 used for constructing transgenic events is used as a negative control; ex vivo bioassay of armyworm resistance for transformation event ZZM 030; the test armyworm is oriental armyworm (Mythimna seperate (Walker)).

The specific identification method is as follows: taking overground parts of fresh corn plants growing to 3-4 and 8-10 leaf stages respectively, bringing the overground parts back to the room, taking tender heart leaves, putting the tender heart leaves into a culture dish, and inoculating 10 instar 1 day of instar; one treatment per dish, each treatment repeated 3 times; the culture was carried out in a climatic chamber at a temperature of 28. + -. 1 ℃ and a light cycle of 14 h (L: D) and a relative humidity of 70-80%. And after 3d, counting the number of the survival larvae and calculating the survival rate of the larvae.

And performing multiple comparative analysis on the survival rates of the armyworms of different varieties, wherein the significance level is 0.05, performing one-time evolution and arcsine transformation on the survival rates before statistical analysis, and comparing the difference significance among treatments. According to the significance of the difference of the survival rates of the larvae of the fed transgenic corn and the control corn, the resistance of the larvae to armyworms is qualitatively judged by referring to the table 5, and the identification result is shown in the table 6. .

TABLE 5. Oriental armyworm in vitro growth evaluation standard

TABLE 6 armyworm resistance in vitro bioassay results for different corn materials

2) Mythimna separata resistance in vitro bioassay experiment II

Taking a Xianzhengda commercial transgenic corn variety Bt11 as a positive control, wherein the material contains Cry1Ab gene and is reported to have armyworm resistance; using a receptor material 249 used for constructing a transgenic event and a conventional corn seedling for raising armyworms in generations in a migratory pest research group of a plant protection research institute of Chinese academy of agricultural sciences as negative controls; in vitro bioassay of armyworm resistance was performed on ZZM 030; the test armyworm is oriental armyworm (Mythimna seperate (Walker)).

The specific identification method is as follows: selecting fresh leaves of 3-4 and 8-10 leaf stages as food for testing armyworm; soaking all the corn leaves in 0.1% sodium hypochlorite solution for 3min for disinfection, washing with distilled water, and air drying; putting a whole corn plant into a 750ml can bottle, inoculating 40 newly hatched larvae into each bottle, and repeating each treatment for 3 times; feeding in artificial climatic box with temperature (24 + -1) ° C, humidity (70 + -5)%, photoperiod 14L; the maize plants of the corresponding treatment were replaced every 2 days and checked for larval mortality on

days

3, 6 and 9 after the start of the test.

Survival data of larvae at 3, 6 and 9 days after different treatments are obtained in the test are processed by Excel, SPSS software (SPSS 16.0) of SPSS Inc., USA is adopted to carry out variance analysis on the survival rates of armyworms of different treatments, and Tukey's HSD is adopted to carry out multiple comparison after the differences are obvious. Survival data were subjected to one evolution and arcsine transformation before statistical analysis.

Table 7 shows the effect of the leaf stage 3-4 of the different materials on the survival rate of the originally hatched armyworms, the data in the table are mean. + -. Standard error, and the same letters in the same column indicate no significant difference at P <0.05 level as determined by Tukey's HSD method. The survival rates of armyworm larvae of the conventional corn seedlings after treatment and 3, 6 and 9 days are over 95 percent, which shows that the larvae used in the test are healthy and the test operation is feasible. The survival rate of larvae hatched at 3-4 leaf stages of different materials is obviously influenced, the survival rate of larvae of each material on the 3 rd day is higher, the survival rate of larvae of Bt11 is obviously lower than that of other materials (P < 0.05), and the survival rate of other treatments is not obviously different (P > 0.05). The survival rate difference of the larvae among the treatments at the 6 th day is more obvious, particularly, the survival rate of the larvae of Bt11 is obviously reduced to only 3l.7%, and the insect resistance level is the resistance according to the evaluation standard of the table 5. The difference in larval survival between treatments was further exacerbated at day 9.

TABLE 7 Effect of different materials at 3-4 leaf stage on survival of initially hatched armyworms

Table 8 shows the effect of different materials at 8-10 leaf stages on the survival rate of the originally hatched armyworms, the data are mean. + -. Standard error, and the same letters in the same column indicate no significant difference at P <0.05 level as determined by Tukey's HSD method. From the larvae survival rate of 3 days after the test, the survival rate of the larvae of Bt11 is significantly lower than that of Xiang 249 and conventional corn seedlings. The difference in survival rates of larvae at day 6 after treatment increased significantly: wherein the survival rate of ZZM030 larvae is less than 45%, showing resistance; xiang 249 and conventional corn seedlings were shown to be highly susceptible. The survival rate of the larvae after 9 days of treatment is high as shown by the results of 249 and the conventional corn seedlings, and is obviously higher than that of other materials; ZZM030 exhibit high resistance.

TABLE 8 Effect of different materials at 8-10 leaf stage on the survival of the armyworm

3) Identification of resistance to armyworm in field

Three test sites are respectively established on Jilin princess mountains, jinan Yin Ma Quan and Yunnan Jinghong, ZZM030 is respectively subjected to field armyworm resistance identification in a living body insect inoculation mode, and the test armyworm is oriental armyworm (Walker) by taking Xiang 249 as a contrast.

The insect inoculation method comprises the following steps: inoculating 40 insects in the 4-6 leaf stage, and inoculating 40 larvae of the first hatched larva in each cell; inoculating the insects twice at an interval of 3 days; after 14 days of the last inoculation, the defoliation grade of each plot inoculated insect strain damaged by armyworm is investigated plant by plant, and the resistance level of the material in the heart-leaf stage is evaluated according to the standard. The classification criteria and resistance level assessment criteria were carried out in accordance with the regulations of Ministry of agriculture 953, publication No. 10.1-2007.

The results are shown in Table 9 and FIG. 5. In table 9, the values are expressed as mean ± standard deviation of 3 replicates, and the difference between different materials in the same spot is significant (P < 0.01) when the lower case letters in the same column are different. In fig. 5, a is transgenic event ZZM030 and B is negative control 249. The edge of the leaf and the missing part of the leaf are wormholes eaten by the armyworm and the silkworm. The average leaf feeding grade of the princess ridge test point ZZM030 is 1.2, the resistance type is high resistance, the average leaf feeding grade of the negative control receptor corn pink 249 is 5.8, and the resistance type is medium resistance; the average leaf eating grade of the Jinan test point ZZM030 is 2.7, the resistance type is resistance, the average leaf eating grade of the negative control receptor corn pink 249 is 5.4, and the resistance type is resistance; the average defoliation rating of the Jinghong test point ZZM030 is 1.5, the resistance type is high resistance, the average defoliation rating of the negative control recipient corn cake 249 is 9.0, and the resistance type is high feeling. The three test points showed significant differences in armyworm resistance between the transformation event ZZM030 and the negative control (recipient corn pink 249), indicating that the transformation event ZZM030 is better resistant to armyworm at the 4-6 leaf stage under field conditions.

TABLE 9 results of field resistance identification of different materials

Example 5 maize transformation event ZZM Southern blot identification

1. Preparation of probes

(1) Preparation of cry1Ab/cry1AcZM Probe

The probe was prepared using pZHZH35006 plasmid DNA as a template. CSP759 (SEQ ID NO 2) and CSP760 (SEQ ID NO 3) are used as primers, a PCR digoxin probe synthesis kit (cat number: 11636090910) of Roche is adopted to synthesize a probe for detecting cry1Ab/cry1AcZM, and the size of the probe is 333bp (the probe sequence is the same as that of S)eq ID No:1, nucleotide sequence shown at positions 6084-6416). The amplification system comprises: 5. Mu.L (50 pg) of DNA template, 0.5. Mu.L of each primer, 5. Mu.L of PCR DIG mix, 0.75. Mu.L of DNA polymerase, 5. Mu.L of PCR buffer (10-fold), ddH ₂ O33.25. Mu.L. The PCR reaction program is: pre-denaturation at 94 ℃ for 5min;35 cycles: denaturation at 94 ℃ 30sec, annealing at 55 ℃ 30sec, extension at 72 ℃ for 45sec; finally, extension was carried out at 72 ℃ for 7min. The effect of the marker amplification was detected using a 1% agarose gel. The specifically amplified amplification product, i.e., the probe used to detect cry1Ab/cry1AcZM, was stored at-20 ℃.

(2) Preparation of the Bar Probe

Using the same method as described in (1), a specific probe for the bar gene was prepared using a primer pair (SEQ ID NO 4 FW-Csp73, SEQ ID NO 5 RV-Csp 74) which was 408bp in length (the probe sequence was identical to the nucleotide sequence shown at positions 1035-1442 of SEQ ID No: 1.

(3) Preparation of cp4Zm Probe

Using the same method as described in (1), a specific probe for the cp4Zm gene was prepared using a primer set (SEQ ID NO 6 FW-Csp1337, SEQ ID NO 7 RV-Csp 1338) and having a length of 1138bp (the probe sequence corresponds to the nucleotide sequence shown at positions 9058-10195 of SEQ ID No: 1).

DNA extraction

Extraction of transgenic maize T ₁ 、T ₂ Or T ₃ And (3) generating leaf genome total DNA of the material, drying the obtained DNA precipitate, dissolving in deionized water, and measuring the concentration for later use.

3. Enzyme digestion, electrophoresis, membrane conversion and development

When the cry1Ab/cry1AcZM probe is used, the corn genomic DNA is digested with HindIII or KpnI in one single digestion.

When using the bar probe, the maize genomic DNA was subjected to a single cleavage with HindIII or EcoRI.

When using the cp4Zm probe, the corn genomic DNA is cleaved with HindIII or KpnI in one enzyme.

Using 200. Mu.L of a restriction enzyme system containing 20. Mu.g of maize genomic DNA, 20. Mu.l of restriction enzyme, 20. Mu.L of 10-fold buffer, add ddH ₂ The volume of O is filled to 200 mu L. Carrying out electrophoresis detection on 20 mu L of the enzyme-digested product after 16hAnd (5) detecting whether the enzyme digestion effect is thorough.

Enzyme digestion product plus ddH ₂ O was replenished to 400. Mu.l, and 1/10 volume of 3M sodium acetate solution (pH 5.2) was added, 4. Mu.L of TaKaRa Dr. GenTLE Precipitation Carrier was added, 2.5-fold volume of absolute ethanol was added, well mixed, and centrifuged at 12,000rpm at 4 ℃ for 15min. The precipitate was washed with 50. Mu.L ddH ₂ O dissolved, and 5. Mu.L of 6-fold loading buffer was added.

The DNA was run through a 0.8% gel at 20V for 16h. Excess lanes and wells were cut off and the remaining gel was treated with denaturing solution 2 times for 15min each time and shaken gently on a shaker. Then treated with neutralization buffer 2 times, each time for 15min, and shaken gently on a shaker. And cleaning once by using ultrapure water. For 10min in 20-fold SSC treatment, membrane transfer was performed for 4 hours or more using a Whatman system.

After the transfer of the membrane was completed, the membrane was placed on Whatman 3MM filter paper soaked with 10 times SSC and crosslinked for 3-5min with an ultraviolet crosslinking instrument. By ddH ₂ And O, simply washing the membrane and drying in the air. The hybridization and development were carried out according to the manual of Roche digoxigenin detection kit I (cat # 11745832910) or Roche digoxigenin detection kit II (cat # 11585614910).

4. Analysis of results

FIG. 6A shows the results of hybridization of transformation event ZZM030 maize genomic DNA to cry1Ab/cry1AcZM specific probe molecules digested with HindIII and KpnI, respectively. The two digestion conditions show a positive band respectively, the band obtained by digestion with HindIII is 10.9kb, the band obtained by digestion with KpnI is 10.5kb, the single copy insertion of the foreign gene cry1Ab/cry1AcZM is shown, and the transformation event is a single copy transformation event.

FIG. 6B shows the results of hybridization of transformation event ZZM030 maize genomic DNA cleaved by HindIII and EcoRI, respectively, with bar specific probe molecules. A positive band is respectively displayed under two enzyme cutting conditions, the band obtained by enzyme cutting with HindIII is 4.2kb, the band obtained by enzyme cutting with EcoRI is 10.0kb, both the band and the band accord with expectation, the single copy insertion of the exogenous gene bar is shown, and the transformation event is a single copy transformation event.

FIG. 6C shows the results of hybridization of transformation event ZZM030 maize genomic DNA cleaved with the cp4Zm specific probe molecule by HindIII and KpnI, respectively. The two digestion conditions show a positive band respectively, the band obtained by HindIII digestion is 10.9kb, the band obtained by KpnI digestion is 10.5kb, both the bands accord with the expectation, the single copy insertion of the exogenous gene cp4Zm is shown, and the transformation event is a single copy transformation event.

Example 6 maize transformation event ZZM030 sequence analysis

In transgenic operations, the same transformation vector is generally used for a large number of genetic transformations, and few excellent transformation events are screened from the many transformation events obtained. Therefore, the detection of the vector, expression element, foreign gene, etc. in the inserted foreign sequence can only prove that the detection sample contains the transgenic component, and different transformation events cannot be distinguished. The different transformation events are characterized by a combination of sequences flanking their insertion site and the inserted foreign sequence. To this end, the flanking sequences of the maize transformation event were isolated and identified in this example.

1. Left flank sequence analysis

Taking the leaf of the plant of the transformation event to be detected to extract the total DNA (T) ₂ Generation or T ₃ Strong transgenic plants grow in generation), and the FPNI-PCR method is utilized to amplify, clone and sequence the flanking sequence of the exogenous gene inserted into the corn genome to obtain a sequence result.

(1) The Tail-PCR primer sequences are shown in Table 10.

TABLE 10 primer sequences

Wherein W = A/T, N = A/G/C/T.

(2) Preparing high-quality corn genome DNA for later use, and diluting to 100 ng/mu L for later use;

(3) The genomic DNA obtained in step (2) is used as a template for the first PCR reaction, and the reaction system is shown in Table 6 below. The reaction procedure is as follows: 95 ℃ for 2.5min;2 following cycles: 94 ℃,10sec,62 ℃,30sec,72 ℃,2min;94 ℃,10sec; at 25 ℃ for 2min;72 ℃ (5.1% ramp), 2min;5 cycles of: 94 ℃,10sec;62 ℃ for 30sec;72 ℃ for 2min;94 ℃,10sec;62 ℃ for 30sec;72 ℃ for 2min;94 ℃,10sec;44 ℃ for 30sec;72 ℃ for 2min;72 ℃ for 5min;20 ℃ for 10min.

TABLE 11 first round PCR reaction System

(4) The first round of PCR product (mixed solution mother liquor) was used as a template for the second round of PCR amplification, and the reaction system is shown in Table 7 below. The reaction procedure is as follows: 94 ℃ for 1.5min; (94 ℃,10sec,62 ℃,30sec,72 ℃,2 min). Times.30 cycles;72 ℃,7min;20 ℃ for 10min.

TABLE 12 second round PCR reaction System

(5) The second round of PCR products (diluted 50 times in the mixture) was used as a template for the third round of PCR amplification, and the reaction system is shown in Table 8 below. The reaction procedure is as follows: 94 ℃ for 1.5min; (94 ℃,10sec,62 ℃,30sec,72 ℃,2 min). Times.30 cycles;72 ℃ for 7min;20 ℃ for 10min.

TABLE 13 third round PCR reaction System

(6) Taking the product of the third round of PCR to carry out electrophoresis detection in 1% (w/v) 1 xTAE agarose gel, and recovering a DNA fragment between 300bp and 2 kb;

(7) The recovered fragments were ligated to T-vector and ligated overnight at 16 ℃;

(8) Converting the ligation product of (7);

(9) With M13F:5'-TGTAAAACGACGGCCAGT-3' and M13R: amplifying the transformation product in the step (8) by a 5'-CAGGAAACAGCTATGACC-3' primer, selecting a positive clone shake culture liquid, and extracting Plasmid DNA by using a TIAnprep Rapid Mini Plasmid Mini Plasmid Kit (centrifugal column type);

(10) Sequencing the plasmid DNA of (9) with a sequencing primer using

Plasmid DNA was sequenced by PCR using Terminator v3.1cycle Sequencing Kit. Sequencing primers are M13F and M13R primers; M13-F:5'-TGTAAAACGACGGCCAGT-3', M13-R:5'-CAGGAAACAGCTATGACC-3';

(11) Purification of PCR products from formamide denaturation by NaAc and absolute ethanol (10)

(12) And (3) starting sequencing the purified and denatured PCR product in the step (11) by using an ABI DNA sequencer 3730 and reading out a sequencing result.

(13) Sequencing results homology searches were performed with the maize genomic sequence in the plantagdb database using the BLASTN tool, with the best matches being the chromosome number and base pair position number of the insertion site, typically 90-100% sequence identity.

(14) The experiment detects that the left flank sequence of the inserted T-DNA is 680bp, and the sequence is shown as SEQ ID NO:1, nucleotides 1-680. The analysis and comparison of the maize B73 whole genome sequence as reference (http:// www.plantgdb.org/ZmGDB/cgi-bin/blastGDB. Pl) confirmed that the insertion point on the left side of the transformation event of the present application is located on chromosome 4 40636901bp.

2. Right flank sequence analysis

Taking the leaf of the plant of the transformation event to be detected to extract the total DNA (T) ₂ Generation or T ₃ Strong transgenic plants grow for generations), and performing amplification, cloning and sequencing on the flanking sequence of the exogenous gene inserted into the corn genome by using a joint PCR method to obtain a sequence result.

(1) Artificially synthesizing primers required by joint PCR, and diluting for later use; the primer sequences are shown in Table 14.

TABLE 14 primer sequences

(2) Preparing high-quality corn genome DNA for later use, and diluting the high-quality corn genome DNA for later use;

(3) By ddH ₂ O, respectively diluting the joint primers AD-L and AD-S to 100 mu mol/L, mixing in equal volume, carrying out water bath denaturation at 94 ℃ for 4min, and naturally cooling to room temperature to obtain a joint of 50 mu mol/L;

(4) The corn genome DNA is cut by enzyme, and the cutting system is as follows:

the enzyme was cleaved at 37 ℃ for 3h.

(5) The connecting joint has the following connecting system:

ligation was performed overnight at 16 ℃.

(6) The genomic DNA restriction-linker ligation product obtained in step (5) was used as a template for the first PCR reaction, and the reaction system is shown in Table 10 below. The reaction procedure is as follows: 94 ℃ for 5min;7 cycles of: 30sec at 94 ℃ and 3min at 72 ℃;32 cycles of: 94 ℃ for 30sec; 3min at 67 ℃; 7min at 67 ℃; 10min at 25 ℃.

TABLE 15 first round PCR reaction System

(7) The first round of PCR products (diluted 40 times in the mixture) was used as a template for the second round of PCR amplification, and the reaction system is shown in Table 11 below. The reaction procedure is as follows: 94 ℃ for 5min;5 cycles of: 94 ℃ for 30sec; 3min at 72 ℃;20 cycles of: 30sec at 94 ℃ and 3min at 67 ℃; 7min at 67 ℃; 10min at 25 ℃.

TABLE 16 second round PCR reaction System

(8) Taking the product of the second round of PCR to carry out electrophoresis detection in 1% (w/v) 1 xTAE agarose gel, and recovering a DNA fragment between 300bp and 2 kb;

(9) The recovered fragments were ligated to T-vector and ligated overnight at 16 ℃;

(10) Converting the ligation product of (9);

(11) Amplifying the transformation product in the step (10) by using M13F and M13R primers, selecting a positive clone shake culture liquid, and extracting Plasmid DNA by using a TIAnprep Rapid Mini Plasmid Kit Rapid Plasmid miniextraction Kit (centrifugal column type);

(12) Sequencing the plasmid DNA of (11) with a sequencing primer using

(13) The PCR product in (12) was denatured with formamide, purified with NaAc and absolute ethanol.

(14) And (3) starting sequencing the purified and denatured PCR product in the step (13) by using an ABI DNA sequencer 3730 and reading out a sequencing result.

(15) Sequencing results a homology search was performed with the maize genomic sequence in the Plant GDB database using the BLASTN tool, with the best match results being the chromosome number and base pair position number of the insertion site, typically 90-100% sequence identity.

(16) The right flanking sequence 460bp of the inserted T-DNA is detected by the experiment and is shown in a sequence SEQ ID NO:1, nucleotides 10915-11374. The right insertion point of the transformation event of the application is determined to be positioned on 40636883bp of chromosome 4 by analysis and comparison by taking a maize B73 whole genome sequence as a reference (http:// www.plantgdb.org/ZmGDB/cgi-bin/blastGDB. Pl).

3. Size of the insert and Effect on the original endogenous genome of Zea mays

The vector size of the exogenous gene single-copy insertion sequence including the left and right border sequences is 10282bp.

The actual size of the insert sequence of the transformation event is 10235bp (681-10915 of SEQ ID NO: 1) determined by a sequencing method of segment PCR amplification exogenous DNA, and the T-DNA insert is 7bp deleted at the left end and 40bp deleted at the right end relative to the expression vector.

The amplification system comprises: 2. Mu.L (200 ng) of DNA template, 0.5. Mu.L of each primer, 0.5. Mu.L of DNA polymerase, 2. Mu.L of PCR buffer (10-fold), ddH ₂ O14.5. Mu.L. The PCR reaction program is: pre-denaturation at 94 ℃ for 5min;35 cycles: denaturation at 94 ℃ 30sec, annealing at 55 ℃ 30sec, extension at 72 ℃ for 3min; finally, extension was carried out at 72 ℃ for 7min. The effect of marker amplification was detected using a 1% agarose gel. Recovering the specifically amplified amplification product, i.e., the target fragment.

The 47bp nucleotide is located in the border sequence of the non-coding region. Deletion of the gene does not affect the integrity of the insect-resistant gene cry1Ab/cry1AcZM and the screening markers bar and cp4Zm genes. DNA sequence analysis and comparison show that the actually inserted nucleotide sequence of the transformation event is completely consistent with the vector sequence, and no base mutation occurs.

In addition, the maize genome at the insertion site is a repetitive sequence, and a 47bp nucleotide deletion does not disrupt any known maize endogenous functional genes.

Example 7 detection of corn transformation event ZZM030

1. Left flank DNA sequence detection:

a pair of primers (SEQ ID NO.8: FW-csp3758 and SEQ ID NO.9: RV-csp 2344) is designed by utilizing the left flank genome sequence of the corn transformation event and the sequence of the 35S polyA terminator in the exogenous fragment, and a qualitative PCR identification method of the transformation event product is established.

The primers designed according to the Left Border (LB) T-DNA 5' end of the exogenous DNA fragment integration site of the maize transformation event ZZM030 are as follows:

seq ID No.8 (FW-csp 3758) 5'-TGATGGTTAATGAGGCAAGA-3' (maize genomic region);

seq ID No.9 (RV-csp 2344): 5'-TATAGGGTTTCGCTCATGTG-3' (35S PolyA region).

The specific primers are used for amplifying DNA fragments at 50-60 ℃ by using temperature gradient PCR to determine the optimal annealing temperature. Results demonstrate that the optimal amplification temperature is 58 ℃; the PCR procedure was 35 cycles at 95 ℃ for 5min, (95 ℃ 30s,50-60 ℃ 30s,72 ℃ 1 min), and 72 ℃ for 7min.

To test the specificity of the primers (FW-csp 3758; RV-csp 2344) for amplification of the transformation event, maize DNA from various sources was used for PCR amplification.

The PCR reaction conditions and procedure were 95 ℃ for 5min,35 cycles below: 30s at 95 deg.C, 30s at 58 deg.C, and 1min at 72 deg.C; 7min at 72 ℃. The results show that only the DNA of the transformation event can have positive results, and other transformation events or negative control maize varieties have negative results, which are shown in FIG. 7A. Wherein lanes 1-4 are sterile water, 249DNA, ZZM030DNA, and other same vector transformation event DNAs, respectively; only ZZM030 genomic DNA lane 3 is clearly visible, the DNA fragment size of 898bp is consistent with the expected size, and the results obtained by clone sequencing of the DNA fragment are also consistent with the expected size.

2. Right flanking DNA sequence detection:

a pair of primers (SEQ ID NO.10: FW-csp3879 and SEQ ID NO.11: RV-csp 3889) is designed by utilizing the cp4Zm sequence in the exogenous fragment and the right flank genome sequence of the corn transformation event, and a qualitative PCR identification method of the transformation event is established.

Primers designed according to the Right Border (RB) T-DNA 5' end of the exogenous DNA fragment integration site of ZZM030 transformation event were:

seq ID No.10 (FW-csp 3879) 5'-AAGATTGAGCTGTCGGATAC-3' (cp 4Zm region),

seq ID No.11 (RV-csp 3889): 5'-TTTGATCATGTGAGGAACGT-3' (maize genomic region).

The optimal annealing temperature is determined by amplifying DNA fragments by using temperature gradient PCR under the condition of 46-61 ℃. Results demonstrate that the optimal amplification temperature is 58 ℃; the PCR reaction program is 95 ℃ for 5min;35 cycles of: 30s at 95 deg.C, 30s at 46-61 deg.C, and 1min at 72 deg.C; 7min at 72 ℃.

To test the specificity of the above primers (FW-csp 3879; RV-csp 3889) for amplification of the transformation event, different sources of maize DNA were used for PCR amplification.

The PCR reaction conditions and procedure were 95 ℃ for 5min;35 cycles of: cycling at 95 ℃ 30s,58 ℃ 30s, and 72 ℃ for 1min; 7min at 72 ℃. The results indicated that only the present transformation event DNA could have a positive result, and that all other transformation events or negative control maize varieties were negative results, see fig. 7B. Wherein lanes 1-4 are sterile water, 249DNA, ZZM030DNA, and other same vector transformation event DNAs, respectively; only ZZM030 genome DNA lane 3 is clearly visible, the size of the DNA fragment 796bp is consistent with the expected size, and the result of clone sequencing of the DNA fragment is also consistent with the expected size.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make various improvements and modifications without departing from the technical principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.

Sequence listing

<110> China seed group Co., ltd

<120> insect-resistant herbicide tolerant corn transformation events

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 11374

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tgatggttaa tgaggcaaga tatgtgaatg gcaccttgaa tggtccactg cccgtctatg 60

agccgcagcc cgttgctctc aaagcaacta gcagcaggga ggcgctacca agcaagttag 120

cacaagtgga ggctgccggg ctcaatgagg atgagatggc gcttatcatc aagcgcttca 180

agaccgcgct aaaaggacgc aaggagtacc ccaacaagaa caagtcaagg ggaaaacgct 240

cctgcttcaa atgcggtaag aatggtcatt ttatagctca atgcctcgat aacgaatgac 300

caggcacaag agaagcatgg gaaaagagag aagaagaaga actaccggaa ggccaagggc 360

gaggcacaca ttgggaagga atgggactcc aactgctcct cctccgactc tgaggatgaa 420

ggactagctg cctcagcctt caacaaatct tcactcttcc ccaacgaacg ccatacatgc 480

cttatggcta aggagaagaa ggtatgtatt cgagacactc ctaagtactc ttcttctagc 540

gatgaggaat cttccgatga tgaggtagat tacactgatt tgtttaagga attatataga 600

gctaaagtag acaaaattaa tgaattaatt gatgctcttg atgaaaaaga taaactacaa 660

gaaaagcaag aggatatttt atatttgtgg tgtaaacaaa ttgacgctta gacaacttaa 720

taacacattg cggacgtttt taatgtactg aattaacgcc gaattaattc gggggatctg 780

gattttagta ctggattttg gttttaggaa ttagaaattt tattgataga agtattttac 840

aaatacaaat acatactaag ggtttcttat atgctcaaca catgagcgaa accctatagg 900

aaccctaatt cccttatctg ggaactactc acacattatt atggagaaac tcgagtcaaa 960

tctcggtgac gggcaggacc ggacggggcg gtaccggcag gctgaagtcc agctgccaga 1020

aacccacgtc atgccagttc ccgtgcttga agccggccgc ccgcagcatg ccgcgggggg 1080

catatccgag cgcctcgtgc atgcgcacgc tcgggtcgtt gggcagcccg atgacagcga 1140

ccacgctctt gaagccctgt gcctccaggg acttcagcag gtgggtgtag agcgtggagc 1200

ccagtcccgt ccgctggtgg cggggggaga cgtacacggt cgactcggcc gtccagtcgt 1260

aggcgttgcg tgccttccag gggcccgcgt aggcgatgcc ggcgacctcg ccgtccacct 1320

cggcgacgag ccagggatag cgctcccgca gacggacgag gtcgtccgtc cactcctgcg 1380

gttcctgcgg ctcggtacgg aagttgaccg tgcttgtctc gatgtagtgg ttgacgatgg 1440

tgcagaccgc cggcatgtcc gcctcggtgg cacggcggat gtcggccggg cgtcgttctg 1500

ggctcatggt agactcgaga gagatagatt tgtagagaga gactggtgat ttcagcgtgt 1560

cctctccaaa tgaaatgaac ttccttatat agaggaaggg tcttgcgaag gatagtggga 1620

ttgtgcgtca tcccttacgt cagtggagat atcacatcaa tccacttgct ttgaagacgt 1680

ggttggaacg tcttcttttt ccacgatgct cctcgtgggt gggggtccat ctttgggacc 1740

actgtcggca gaggcatctt gaacgatagc ctttccttta tcgcaatgat ggcatttgta 1800

ggtgccacct tccttttcta ctgtcctttt gatgaagtga cagatagctg ggcaatggaa 1860

tccgaggagg tttcccgata ttaccctttg ttgaaaagtc tcaatagccc tttggtcttc 1920

tgagactgta tctttgatat tcttggagta gacgagagtg tcgtgctcca ccatgttcac 1980

atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg atgctcctcg 2040

tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaacg atagcctttc 2100

ctttatcgca atgatggcat ttgtaggtgc caccttcctt ttctactgtc cttttgatga 2160

agtgacagat agctgggcaa tggaatccga ggaggtttcc cgatattacc ctttgttgaa 2220

aagtctcaat agccctttgg tcttctgaga ctgtatcttt gatattcttg gagtagacga 2280

gagtgtcgtg ctccaccatg ttggcaagct gctctagcca atacgcaaac cgcctctccc 2340

cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg 2400

cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca 2460

ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg 2520

aaacagctat gacatgatta cgaattcgag ctcggtaccc ggggatcctc tagagtcgac 2580

ctgcaggcat gcaagcttat ccagcttgca tgcctgcagt gcagcgtgac ccggtcgtgc 2640

ccctctctag agataatgag cattgcatgt ctaagttata aaaaattacc acatattttt 2700

tttgtcacac ttgtttgaag tgcagtttat ctatctttat acatatattt aaactttact 2760

ctacgaataa tataatctat agtactacaa taatatcagt gttttagaga atcatataaa 2820

tgaacagtta gacatggtct aaaggacaat tgagtatttt gacaacagga ctctacagtt 2880

ttatcttttt agtgtgcatg tgttctcctt tttttttgca aatagcttca cctatataat 2940

acttcatcca ttttattagt acatccattt agggtttagg gttaatggtt tttatagact 3000

aattttttta gtacatctat tttattctat tttagcctct aaattaagaa aactaaaact 3060

ctattttagt ttttttattt aataatttag atataaaata gaataaaata aagtgactaa 3120

aaattaaaca aatacccttt aagaaattaa aaaaactaag gaaacatttt tcttgtttcg 3180

agtagataat gccagcctgt taaacgccgt cgacgagtct aacggacacc aaccagcgaa 3240

ccagcagcgt cgcgtcgggc caagcgaagc agacggcacg gcatctctgt cgctgcctct 3300

ggacccctct cgagagttcc gctccaccgt tggacttgct ccgctgtcgg catccagaaa 3360

ttgcgtggcg gagcggcaga cgtgagccgg cacggcaggc ggcctcctcc tcctctcacg 3420

gcaccggcag ctacggggga ttcctttccc accgctcctt cgctttccct tcctcgcccg 3480

ccgtaataaa tagacacccc ctccacaccc tctttcccca acctcgtgtt gttcggagcg 3540

cacacacaca caaccagatc tcccccaaat ccacccgtcg gcacctccgc ttcaaggtac 3600

gccgctcgtc ctcccccccc ccctctctac cttctctaga tcggcgttcc ggtccatggt 3660

tagggcccgg tagttctact tctgttcatg tttgtgttag atccgtgttt gtgttagatc 3720

cgtgctgcta gcgttcgtac acggatgcga cctgtacgtc agacacgttc tgattgctaa 3780

cttgccagtg tttctctttg gggaatcctg ggatggctct agccgttccg cagacgggat 3840

cgatttcatg attttttttg tttcgttgca tagggtttgg tttgcccttt tcctttattt 3900

caatatatgc cgtgcacttg tttgtcgggt catcttttca tgcttttttt tgtcttggtt 3960

gtgatgatgt ggtctggttg ggcggtcgtt ctagatcgga gtagaattct gtttcaaact 4020

acctggtgga tttattaatt ttggatctgt atgtgtgtgc catacatatt catagttacg 4080

aattgaagat gatggatgga aatatcgatc taggataggt atacatgttg atgcgggttt 4140

tactgatgca tatacagaga tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg 4200

ggcggtcgtt cattcgttct agatcggagt agaatactgt ttcaaactac ctggtgtatt 4260

tattaatttt ggaactgtat gtgtgtgtca tacatcttca tagttacgag tttaagatgg 4320

atggaaatat cgatctagga taggtataca tgttgatgtg ggttttactg atgcatatac 4380

atgatggcat atgcagcatc tattcatatg ctctaacctt gagtacctat ctattataat 4440

aaacaagtat gttttataat tattttgatc ttgatatact tggatgatgg catatgcagc 4500

agctatatgt ggattttttt agccctgcct tcatacgcta tttatttgct tggtactgtt 4560

tcttttgtcg atgctcaccc tgttgtttgg tgttacttct gcaggtcgac tctagaggat 4620

cgtattttta caacaattac caacaacaac aaacaacaaa caacattaca attactattt 4680

acaattacaa ccatggattg ccggccctac aactgcctgt cgaaccctga ggtggaggtc 4740

ctgggcggcg agcggattga gactggctac acaccgattg acatctcact ctccctgacc 4800

cagttcctcc tgtcggagtt cgtgccaggc gctgggttcg ttctcggcct ggtggatatc 4860

atttggggca tcttcgggcc aagccagtgg gacgctttcc tggtccagat cgagcagctc 4920

attaatcaga ggatcgagga gttcgcgcgg aaccaggcta ttagccgcct cgagggcctg 4980

tcgaacctct accagatcta cgccgagagc ttcagggagt gggaggctga tccgacgaac 5040

cccgccctga gggaggagat gcggattcag ttcaatgaca tgaactccgc tctgaccacg 5100

gctatcccac tcttcgcggt gcagaattac caggtcccac tcctgagcgt ctacgtgcag 5160

gctgcgaacc tccacctgtc tgtgctgcgc gatgtttcag tgttcggcca gacctggggg 5220

ttcgacgctg ctacgattaa ttccaggtac aacgatctga cacggctcat cggcaattac 5280

actgaccatg ccgttcggtg gtacaacacc ggcctcgaga gggtgtgggg gccagactcc 5340

agggattgga ttaggtacaa ccagttccgc agggagctca cactgactgt cctggacatc 5400

gtttccctct tcccaaacta cgatagccgg acctacccta ttcgcacggt gtcccagctg 5460

acaagggaga tctacactaa tccagtcctc gagaacttcg acggctcttt ccgcgggtca 5520

gctcagggca ttgaggggtc catcaggagc cctcacctga tggatatcct caactcaatc 5580

accatctaca cggacgctca ccgcggcgag tactactggt ccgggcatca gatcatggct 5640

tccccagtcg gcttcagcgg gccagagttc accttcccac tgtacggcac gatggggaac 5700

gctgctccac agcagaggat cgttgctcag ctcggccagg gggtgtaccg cacactgtcc 5760

agcactctct accggcgccc gttcaacatc ggcattaaca atcagcagct gagcgtgctc 5820

gacggcacag agttcgccta cgggacttcg tctaacctgc cctcggcggt ctacaggaag 5880

tcgggcaccg ttgactctct cgatgagatc ccgccccaga acaataacgt cccacctcgc 5940

cagggcttct cgcacaggct gtcgcatgtt tctatgttcc ggtcaggctt ctccaactca 6000

tccgtctcca tcattagggc cccgatgttc tcatggatcc accggtccgc ggagttcaat 6060

aacatcattg ctagcgattc gatcacgcag attccagcgg tcaagggcaa tttcctcttc 6120

aacgggagcg ttatctcggg ccctgggttc acaggcgggg acctggtgag gctcaatagc 6180

tcgggcaata acatccagaa caggcggtac attgaggtcc caatccactt cccttctacc 6240

tcaacgcgct acagggtccg ggttcgctac gcgtccgtga caccaattca tctgaatgtc 6300

aactggggca attcttcaat cttctcgaac actgtgcctg ccacagcgac ttctctggac 6360

aatctccagt ccagcgattt cggctacttc gagtctgcta acgccttcac ctcgtctctc 6420

ggcaatatcg tgggggtccg caacttcagc ggcacggctg gcgttattat tgataggttc 6480

gagttcatcc ctgttactgc taccctggag gctgagtaag taggtgagga attctttgag 6540

tattatggca ttggaaaagc cattgttctg cttgtaattt actgtgttct ttcagttttt 6600

gttttcggac atcaaaaaaa aaaaaaaaaa aaaaaaaaaa tttaacaaaa aaaaaaaaaa 6660

aaaaaaaaaa gtttaattcg attatcctcg agcgaatttc cccgatcgtt caaacatttg 6720

gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt 6780

tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag 6840

atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat 6900

atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcgggt 6960

ttagcttgaa ttcattatgt ggtctaggta ggtctatata taatgtcagt ctcagtgggg 7020

atttcatgtc cctgttacca atgcacccat attttggaaa caatgtaaaa agagttttat 7080

ccccataaaa ctctctctac tcccatgaaa cttttatcat ctctctattc atcaatacgg 7140

tgtcacatca gcctatttaa tgcgtttaaa actctgatga aaccccactt agactggcct 7200

cagaaaactt gaaatgttct aaaaaaattc aagcccatgc atgattgaag caaacggtat 7260

agcaacggtg ttaacctggt ctagtgatct cttgtaatcc ttaacggcca cctaccgcag 7320

gtagcaaacg gcgtccccct cctcgatatc tccgcggcgg cctctggctt tttccgcgga 7380

attgcgcggt ggggacggat tccacgagac cgcgacgcaa ccgcctctcg ccgctgggcc 7440

ccacaccgct cggtgccgta gcctcacggg actctttctc cctcctcccc cgttataaat 7500

tggcttcatc ccctcccggc ctcatccatc caaatcccag tccccaatcc cagcccatcg 7560

tcggagaaat tcatcgaagc gaagcgaatc ctcgcgatcc tctcaaggta ctgcgagttt 7620

tcgatccccc tctcgacccc tcgtatgttt gtgttcgtcg tagcgtttga ttaggtatgc 7680

tttccctgtt tgtgttcgtc gtagcgtttg attaggtatg ctttccctgt tcgtgttcat 7740

cgtagtgttt gattaggtcg tgtgaggcga tggcctgcta gcgtccttcg atctgtagtc 7800

gatttgcggg tcgtggtgta gatctgcggg ccgtgatgaa gttatttggt gtgatcgtgc 7860

tcgcctgatt ctgcgggttg gctcgagtag atatgatggt tggaccggtt ggttcgttta 7920

ccgcgctagg gttgggctgg gatgatgttg catgcgccgt tgcgcgtgat cccgcggtag 7980

gacttgcgtt tgattgccag atctcgttac gattatgtga tttggtttgg actttttaga 8040

tctgtagctt ctgcttatgt gccagatgcg cctactgctc atatgcctga tgataatcat 8100

aaatggctgt ggaactatgt atcagctaca ggtgtaggga cttgcgtcta attgtttggt 8160

cctgtactca tgttgcaatt atgcgattta gtttaggttg tttgttccac tcatctaggc 8220

tgtaaaaggg acactgctta gattgctgtt taatcttttt agtagattat attatattgg 8280

taacttatta cccttattac atgccatacg tgacttctgc tcatgcctga tgataatcat 8340

agatcactgt ggaattaatt agttgattgt tgaatcatgt ttcatgtaca taccatggca 8400

caattgctta gttccttaac aaatgcaaat tttactgatc catgtatgat ttgcgtggtt 8460

ctctaatgtg aaatactata gctacttgtt agtaagaatc aggttcgtat gcttaatgct 8520

gtatgtgcct tctgctcatg cctgatgata atcatatatc actggaatta attagttgat 8580

cgtttaatca tatatcaagt acataccatg gcacaatttt tagtcactta acccatgcag 8640

attgaactgg tccctgcatg ttttgctaaa ttgttctatt ctgattagac catatatcat 8700

gtattttttt tttggtaatg gttctcttat tttaaatgct atatagttct ggtacttgtt 8760

agaaagatct gcttcatagt ttagttgcct atccctcgaa ttaggatgct gagcagctga 8820

tcctatagct ttgtttcatg tatcaattct tttgtgttca acagtcagtt tttgttagat 8880

tcattgtaac ttatggtcgc ttactcttct ggtcctcaat gcttgcagct gcaggtcgac 8940

tctagaggat ccatttttac aacaattacc aacaacaaca aacaacaaac aacattacaa 9000

ttacatttac aattaccatg gctcagattc gcagcatggc tcagggcatt cagacactct 9060

cgctcaactc gtccaacctc agcaagactc agaaggggcc gctcgtgtcc aacagcctgt 9120

tcttcggctc gaagaagctc acgcagatca gcgcgaagtc gctgggcgtg ttcaagaagg 9180

acagcgtcct ccgcgtggtc aggaagtcca gcttccggat ctcggcttct gtggctaccg 9240

cggaggctca cggcgcctcg tctcgcccag ctaccgctag gaagtcatcc gggctgagcg 9300

gcacggtccg catccctggc gacaagtcaa tttcccatag gtcattcatg ttcggcgggc 9360

tcgcttccgg cgagacaagg atcactgggc tcctggaggg cgaggacgtg attaacacgg 9420

ggaaggctat gcaggcgatg ggcgctcgca tcaggaagga gggggacaca tggatcattg 9480

atggcgtcgg gaacggcggg ctcctggctc cagaggctcc tctggacttc gggaatgctg 9540

ctacaggctg ccgcctgact atggggctcg tcggcgttta cgacttcgat tcgacattca 9600

tcggcgatgc ctctctcact aagaggccaa tgggccgggt gctgaaccct ctcagggaga 9660

tgggcgtgca ggtcaagtcc gaggacgggg ataggctgcc agttaccctc aggggcccaa 9720

agacaccaac tccaatcacg taccgggtcc cgatggcttc cgctcaggtt aagagcgcgg 9780

tgctcctggc tgggctgaac accccgggca tcaccacggt catcgagccc attatgacac 9840

gcgaccacac tgagaagatg ctccagggct tcggggcgaa tctcaccgtt gagacggacg 9900

ctgatggcgt gcggacaatc cgcctggagg gcagggggaa gctcactggc caggtcatcg 9960

acgtcccagg cgacccgtcc tccaccgctt tcccactggt ggctgctctc ctggtccctg 10020

gctccgacgt tactatcctg aacgtgctca tgaatccgac ccggacgggc ctcattctga 10080

ccctccagga gatgggcgcc gatatcgagg tcatcaaccc aaggctcgct ggcggggagg 10140

acgtcgccga tctgcgggtt cgctcttcaa ccctcaaggg cgttacggtg ccagaggaca 10200

gggctccttc catgatcgat gagtacccaa ttctggctgt cgcggctgcc ttcgctgagg 10260

gggccacggt catgaatggc ctggaggagc tgagggttaa ggagtctgac cggctctcag 10320

cggtggctaa cgggctgaag ctcaatggcg tggactgcga tgagggcgag acctctctgg 10380

ttgtgagggg gcggccggac ggcaaggggc tcggcaacgc tagcggcgcg gctgtggcta 10440

ctcacctcga tcataggatc gccatgagct tcctggtcat gggcctcgtt tcggagaatc 10500

cggtcacagt tgacgatgcc accatgattg cgacgtcctt ccccgagttc atggacctga 10560

tggctgggct gggggcgaag attgagctgt cggataccaa ggctgcgtga gagctcatcg 10620

aattccgaat ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc 10680

tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat 10740

aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca 10800

attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc 10860

gcgcgcggtg tcatctatgt tactagatcg ggtttaaact atcagtgttt gacaagtttg 10920

ttagtgttca aaaatctctt gctttagaaa ttaagaaaaa tgaaatattg tcatctgagt 10980

tatcttcctg tcatgaatct attgctagct taaaggattt aaataatgat ttgaatacta 11040

agttagaaaa agcaaatgca actagttcat gtgtagaaca cgtagttatt tgcaatagat 11100

gtaaagatgt taattttgat gaacatgctg ctactattgc taagttaaat aatgatgttg 11160

caagtcttaa tgatcaattt aagacttgca aaaatgatta tgagaaatta aaatttgcta 11220

gggatgccta caccgttggt agacacccct caattaaaaa tgaacttggt tttcgaaagg 11280

aaaccaagaa cttaacaagc caaaggactt ccgatctcaa ggggaaaggg aaggctccta 11340

tggtcagtag ctcacgttcc tcacatgatc aaaa 11374

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cacgcagatt ccagcggtca a 21

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gacgaggtga aggcgttagc a 21

<210> 4

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cagttcccgt gcttgaag 18

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

caccatcgtc aaccactac 19

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tctcgctcaa ctcgtccaac ct 22

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tctggcaccg taacgccctt ga 22

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tgatggttaa tgaggcaaga 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tatagggttt cgctcatgtg 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aagattgagc tgtcggatac 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tttgatcatg tgaggaacgt 20

Claims

1. 1 or a completely complementary sequence thereof.

2. The nucleic acid molecule of claim 1, comprising the following expression cassettes:

a first expression cassette for expressing a glufosinate-resistant gene, which has a sequence shown as 748-2288 th nucleotides of SEQ ID NO. 1;

a second expression cassette for expressing insect-resistant genes, which has a sequence shown as 2620-6959 nucleotides of SEQ ID NO. 1; and

a third expression cassette for expressing glyphosate-resistant gene, which has a sequence shown as 6968-10892 th nucleotides of SEQ ID NO. 1.

3. The nucleic acid molecule of claim 1, obtained by introducing into the genome of maize:

a third expression cassette for expressing glyphosate-resistant gene, which has a sequence shown as nucleotide 69668-10892 of SEQ ID NO. 1.

4. A primer pair for detecting a corn transformation event, the primer pair being:

nucleotide sequences shown as SEQ ID NO.8 and SEQ ID NO. 9; and

the nucleotide sequences shown as SEQ ID NO.10 and SEQ ID NO.11,

wherein the maize transformation event is the sequence shown in SEQ ID NO 1.

5. A kit for detecting a corn transformation event comprising the primer pair of claim 4.

6. A microarray for detecting corn transformation events comprising the primer pair of claim 4.

7. A method of detecting a corn transformation event comprising detecting the presence of the transformation event in a test sample using the primer pair of claim 4, the kit of claim 5, or the microarray of claim 6.

8. A method of breeding maize, the method comprising the steps of:

1) Obtaining maize comprising the nucleic acid molecule of claim 1; and

2) Subjecting the corn obtained in step 1) to pollen culture, unfertilized embryo culture, double culture, selfing or hybridization or a combination thereof to obtain a corn plant or plant part.

9. A method of breeding maize, the method comprising the steps of:

1) Obtaining maize comprising the nucleic acid molecule of claim 1; and

2) Subjecting the corn obtained in step 1) to cell culture, tissue culture or a combination thereof to obtain a corn plant or plant part.

10. The method of claim 8 or 9, wherein the method further comprises:

3) Identifying the maize plants obtained in step 2) for resistance to the herbicides glufosinate and glyphosate and borer and/or armyworm and detecting the presence or absence of the nucleic acid molecule therein using the method of claim 7.

11. A method of controlling a lepidopteran pest population comprising contacting said lepidopteran pest population with a corn plant or plant part obtained by the method of claim 10, wherein said lepidopteran pest is asian corn borer (Ostrinia furnacalis), european corn borer (Ostrinia nubilalis), or oriental armyworm (Mythimna separate).

12. A method of killing a lepidopteran pest, comprising contacting said lepidopteran pest with a pesticidally-effective amount of a corn plant or plant part obtained by the method of claim 10, wherein said lepidopteran pest is asian corn borer (Ostrinia furnacalis), european corn borer (Ostrinia nubilalis), or oriental armyworm (Mythimna separate).